ARTICLE
Received 20 Aug 2015 | Accepted 18 Feb 2016 | Published 29 Mar 2016 DOI: 10.1038/ncomms11097 OPEN Defects in TRPM7 channel function deregulate thrombopoiesis through altered cellular Mg2 þ homeostasis and cytoskeletal architecture
Simon Stritt1,2, Paquita Nurden1,3, Remi Favier4,5, Marie Favier5,6,7,8, Silvia Ferioli9, Sanjeev K. Gotru1,2, Judith M.M. van Eeuwijk1,2, Harald Schulze1, Alan T. Nurden3, Michele P. Lambert10,11, Ernest Turro12,13,14,15, Stephanie Burger-Stritt16, Masayuki Matsushita17, Lorenz Mittermeier9, Paola Ballerini4, Susanna Zierler9, Michael A. Laffan18,19, Vladimir Chubanov9, Thomas Gudermann9,20,21, Bernhard Nieswandt1,2 & Attila Braun1,2
Mg2 þ plays a vital role in platelet function, but despite implications for life-threatening 2 þ conditions such as stroke or myocardial infarction, the mechanisms controlling [Mg ]i in megakaryocytes (MKs) and platelets are largely unknown. Transient receptor potential melastatin-like 7 channel (TRPM7) is a ubiquitous, constitutively active cation channel with a cytosolic a-kinase domain that is critical for embryonic development and cell survival. Here we report that impaired channel function of TRPM7 in MKs causes macrothrombocytopenia in mice (Trpm7fl/fl-Pf4Cre) and likely in several members of a human pedigree that, in addition, suffer from atrial fibrillation. The defect in platelet biogenesis is mainly caused by cytoskeletal alterations resulting in impaired proplatelet formation by Trpm7fl/fl-Pf4Cre MKs, which is rescued by Mg2 þ supplementation or chemical inhibition of non-muscle myosin IIA heavy chain activity. Collectively, our findings reveal that TRPM7 dysfunction may cause macrothrombocytopenia in humans and mice.
1 Chair of Experimental Biomedicine, University Hospital, University of Wu¨rzburg, Josef-Schneider-Strasse 2, 97078 Wu¨rzburg, Germany. 2 Rudolf Virchow Centre, University of Wu¨rzburg, Josef-Schneider-Strasse 2, 97078 Wu¨rzburg, Germany. 3 Institut Hospitalo-Universitaire LIRYC, Plateforme Technologique d’Innovation Biome´dicale, Hoˆpital Xavier Arnozan, Avenue du Haut Le´veˆque, 33604 Pessac, France. 4 Assistance Publique—Hoˆpitaux de Paris, Haematological Laboratory, Armand Trousseau Children Hospital, 26 Avenue du Docteur Arnold-Netter, 75012 Paris, France. 5 Inserm U1170, Gustave Roussy, University Paris Sud, 114 Rue Edouard Vaillant, 94805 Villejuif, France. 6 Inra, UMR_INRA 1260, 27 Boulevard Jean Moulin, 13385 Marseille, France. 7 Aix Marseille Universite´, 58 Boulevard Charles Livon, 13284 Marseille, France. 8 Inserm UMR_S 1062, 27 Boulevard Jean Moulin, 13385 Marseille, France. 9 Walther-Straub-Institute for Pharmacology and Toxicology, Ludwig-Maximilians University Munich, Goethestrae 33, 80336 Munich, Germany. 10 Division of Haematology, Children’s Hospital of Philadelphia, 3401 Civic Center Blvd, Philadelphia, Pennsylvania 19104, USA. 11 Department of Paediatrics, Perelman School of Medicine at the University of Pennsylvania, 3400 Civic Center Blvd, Philadelphia, Pennsylvania 19104, USA. 12 Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Francis Crick Ave, Cambridge CB2 0S, UK. 13 NHS Blood and Transplant, Cambridge Biomedical Campus, Francis Crick Ave, Cambridge CB2 0S, UK. 14 Medical Research Council Biostatistics Unit, Cambridge Institute of Public Health, Cambridge Biomedical Campus, Robinson Way, Cambridge CB2 0SR, UK. 15 NIHR BioResource—Rare Diseases, Cambridge University Hospitals, Cambridge Biomedical Campus, Hills Rd, Cambridge CB2 0QQ, UK. 16 Endocrinology and Diabetes Unit, Department of Internal Medicine I, University Hospital of Wu¨rzburg, Oberdu¨rrbacher Strasse 6, 97080 Wu¨rzburg, Germany. 17 Department of Molecular & Cellular Physiology, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Okinawa 903-0215, Japan. 18 Centre for Haematology, Hammersmith Campus, Imperial College Academic Health Sciences Centre, Imperial College London, Du Cane Road, London SW7 2AZ, UK. 19 Imperial College Healthcare NHS Trust, Du Cane Road, London SW7 2AZ, UK. 20 Comprehensive Pneumology Center Munich (CPC-M), German Center for Lung Research, Max-Lebsche-Platz 31, 81377 Munich, Germany. 21 DZHK (German Centre for Cardiovascular Research), Munich Heart Alliance, Lazarettstrae 36, 80636 Munich, Germany. Correspondence and requests for materials should be addressed to B.N. (email: [email protected]) or to A.B. (email: [email protected]).
NATURE COMMUNICATIONS | 7:11097 | DOI: 10.1038/ncomms11097 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11097
latelets are continuously produced from megakaryocytes alterations due to increased actomyosin contractility and can be (MKs) in the bone marrow by a cytoskeleton-driven rescued by either Mg2 þ supplementation or chemical inhibition Pprocess of which the molecular regulation is not fully of NMMIIA activity. Collectively, our findings reveal TRPM7 understood. MKs extend long cytoplasmic protrusions into bone dysfunction as a novel cause of macrothrombocytopenia in mice marrow sinusoids, where larger fragments, so-called preplatelets, and potentially in humans too. are shed and further divide within the circulation to give rise to platelets (Supplementary Movie 1)1–3. Results Transient receptor potential melastatin-like 7 (TRPM7) Defects in TRPM7 cause macrothrombocytopenia.We channel and kinase domain, but not its kinase activity, are identified TRPM7 as the key Mg2 þ channel and magnesium critical for embryonic development4–6 and knockdown or transporter 1 (MagT1) to be expressed in murine platelets cell-specific TRPM7 knockout approaches give rise to (Supplementary Fig. 1a) and generated MK- and impaired cytoskeletal organization, cell migration, proliferation, platelet-specific TRPM7 knockout mice (Supplementary polarization and survival. These defects could partially be Fig. 1b,c). The absence of TRPM7 currents in patch clamp explained by increased non-muscle myosin IIA heavy chain measurements confirmed the efficacy of the targeting strategy (NMMIIA)-mediated contractility of the actin cytoskeleton5,7–13. in primary bone marrow MKs (Supplementary Fig. 1d). Of note, among other substrates, the kinase domain of TRPM7 Unexpectedly, these mice displayed a macrothrombocytopenia phosphorylates annexin I and NMMIIA, thus interfering with (Fig. 1a,b) with enlarged and spherical platelets, often containing cell survival and cytoskeletal rearrangements14,15. Interestingly, large vacuoles as revealed by electron microscopy (Fig. 1c). several variants of NMMIIA similarly altered the contractility In contrast, mice carrying a kinase-dead K1646R mutation of the actomyosin complex in MKs, thereby interfering with in Trpm7 (ref. 6; Trpm7KI) showed normal platelet counts, size proplatelet formation in humans and mice16. During mega- and morphology, thus suggesting that the lack of TRPM7 channel karyopoiesis, NMMIIA activity is suppressed by phosphorylation function rather than its kinase activity accounts for the of its C-terminus, enabling MK polyploidisation and ultimately macrothrombocytopenia in the mutant mice (Supplementary proplatelet formation17. However, for proper platelet fission and Fig. 2a–e). In line with this notion, intracellular Mg2 þ sizing, NMMIIA needs to be re-activated under shear stress in the concentrations in Trpm7fl/fl-Pf4Cre platelets, but not in Trpm7KI circulation16,18. Although both kinase and channel activity of platelets, were decreased (Fig. 1d; Supplementary Fig. 2f)6. TRPM7 have been proposed to regulate cytoskeletal dynamics, channel activity alone was sufficient to restore cell polarization, 10,13,19 morphology and migration , suggesting a critical role of Impaired proplatelet formation causes thrombocytopenia. cations therein. Consequently, the differential role of TRPM7 A mildly reduced platelet lifespan in Trpm7fl/fl-Pf4Cre mice channel versus kinase activity in the regulation of the (t½ ¼ 43.6 h for wild type (WT) versus t½ ¼ 35.7 h for cytoskeleton still remains unclear. Moreover, TRPM7 has been Trpm7fl/fl-Pf4Cre mice), however, was insufficient to explain the implicated as a key regulator of signal conductance in the murine reduced platelet count and was not associated with altered heart by regulating the expression of different pacemaker platelet terminal galactose levels (Supplementary Fig. 3a,b). channels, such as HCN4 (ref. 20). Although TRPM7-mediated Immunostaining of whole-femora bone marrow sections (Fig. 2a) cation influx has been detected in MKs (ref. 21), its role in revealed an increased number of MKs in the mutant mice thrombopoiesis has not been investigated to date. (6.3±0.3 for WT versus 13.3±1.6 for Trpm7fl/fl-Pf4Cre mice; Here we report that impaired channel function but not kinase Fig. 2b). The MKs in Trpm7fl/fl-Pf4Cre mice were also located activity of TRPM7 in MKs causes macrothrombocytopenia further from bone marrow sinusoids than in controls (Fig. 2c) fl/fl-Pf4Cre in Trpm7 mice and likely in several members of a suggesting impaired migration of MK-precursors to bone human pedigree, which, in addition, feature atrial fibrillation. marrow sinusoids. Although splenomegaly was not observed in The impaired proplatelet formation is associated with cytoskeletal Trpm7fl/fl-Pf4Cre mice, we found an increased number of MKs in
a b c V 1.0 *** 7 *** V 6
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Figure 1 | TRPM7 deficiency causes mild macrothrombocytopenia in mice. Moderately reduced peripheral platelet counts (a) and significantly increased platelet volume (b) were quantified with an automated cell analyser. Values are mean±s.d. (n ¼ 7). (c) Transmission electron microscopy (TEM) analysis shows discoid WT platelets, but enlarged and round resting platelets of Trpm7fl/fl-Pf4Cre mice. V, vacuole. Scale bar, 1 mm. (d) Total platelet cation content was determined by inductively coupled plasma mass spectrometry. Values are mean±s.d. (n ¼ 5). Unpaired Student’s t-test; ***Po0.001; **Po0.01; *Po0.05.
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a b c d 16 *** 50 30 ** 40 *** 25 12 m) μ 30 20 8 20 15 WT 10 10 4 Distance to sinusoid ( 5 0 Mean ploidy (N) MK per visualMK per field 0 0 CD105 GPIbα DAPI e g 40 *** 1.2 *** ) *** 30 –1 WT 0.8 Trpm7 fl/fl-Pf4Cre fl/fl-Pf4Cre 20 0.4 10 Trpm7 PPF MK (%) MKs (% min 0 0 PPF BPF h
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Figure 2 | Cytoskeletal alterations account for the macrothrombocytopenia. (a) Confocal microscopy images of immunostained bone marrow sections. Scale bars, 50 mm (left panel. Scale bars, 15 mm (right panel). MKs, proplatelets and platelets are identified by GPIb staining (green). Endoglin staining (red) labels vessels; 40,6-diamidino-2-phenylindole (DAPI) stains nuclei (blue). (b) Quantification of bone marrow MKs per visual field (328 Â 246 mm). Values are mean±s.d. (n ¼ 6; each 20 visual fields were analysed). (c) MK localization was quantified as their distance from bone marrow sinusoids. Values are mean±s.d. (n ¼ 6; 300 MKs). Wilcoxon–Mann–Whitney test; ***Po0.001. (d) Mean ploidy of primary bone marrow MKs. Values are mean±s.d. (n ¼ 6). (e) Percentage of proplatelet-forming foetal liver-derived MKs in vitro on day 4 of culture. Values are mean±s.d. (n ¼ 7). (f) Intravital two-photon microscopy of bone marrow MKs in the skulls of WT and Trpm7fl/fl-Pf4Cre mice. Arrow shows a normal sized proplatelet in a WT bone marrow sinusoid (PPF); dashed arrow indicates bulky proplatelet in the sinus of a mutant mouse (BPF). Rhodamine dextran labels vessels (red) and anti-GPIX antibodies label MKs and platelets (green). Scale bars, 25 mm. (g) Quantification of proplatelet formation in vivo by intravital two-photon microscopy. Proplatelet formation was quantified during a period of 13 h (27 movies each 30 min duration) in WT and 6.5 h (13 movies each 30 min duration) in Trpm7fl/fl-Pf4Cre mice and normalized to the cell number per visual observed field. Values are mean±s.d. (n ¼ 12 versus 6). (h) Confocal microscopy of in vitro cultured foetal liver-derived MKs revealed an increased content of microtubules in rarely found and less branched proplatelets of Trpm7fl/fl-Pf4Cre MKs. a-tubulin (green) and F-actin (red) stain the cytoskeleton. DAPI, blue. Scale bar, 25 mm. (i,j) Transmission electron microscopy (TEM) analysis of bone marrow MKs. Trpm7fl/fl-Pf4Cre MKs display an irregular distribution of granules, tortuous membranes (i) and proplatelet structure (j). EC, endothelial cell; VS, vascular sinusoid; RBC, red blood cell. Scale bars, 2.5 mm. All images are representative of at least five animals. Unpaired Student’s t-test (if not stated otherwise); ***Po0.001; **Po0.01. an expanded red pulp in the spleen and in line with the increased distant localization from bone marrow sinusoids (Fig. 2c). MK numbers, plasma thrombopoietin levels were decreased, thus Interestingly, mutant MKs displayed an increased mean ploidy further indicating deregulated megakaryopoiesis (Supplementary as compared with controls (16.9 N±1.6 N in controls versus Fig. 4a–d). 22.4 N±3.4 N for Trpm7fl/fl-Pf4Cre mice; Fig. 2d) thus excluding In contrast to a previous report on a neuroblastoma cell line22, impaired MK maturation as the cause of thrombocytopenia. the formation of podosomes, F-actin rich structures that are Despite the increased ploidy in vivo, we found a decreased thought to serve cell migration and proplatelet protrusion23, was proplatelet formation for both foetal liver- (Fig. 2e) and bone unaltered in Trpm7fl/fl-Pf4Cre MKs (Supplementary Fig. 5a) marrow-derived (Supplementary Fig. 5b) Trpm7fl/fl-Pf4Cre suggesting that other defects must account for their more MKs in vitro. This was further confirmed in vivo by intravital
NATURE COMMUNICATIONS | 7:11097 | DOI: 10.1038/ncomms11097 | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11097 two-photon microscopy of the bone marrow (0.88% min À 1 was also altered in Trpm7fl/fl-Pf4Cre platelets. However, in contrast ±0.16% min À 1 of WT MKs formed proplatelets versus to MKs, NMMIIA predominated in the cell cortex of WT 0.23% min À 1±0.12% min À 1 of Trpm7fl/fl-Pf4Cre MKs), which platelets reminiscent of the marginal band, whereas it was revealed that Trpm7fl/fl-Pf4Cre MKs preferentially formed short homogeneously distributed in platelets of Trpm7fl/fl-Pf4Cre mice and bulky proplatelet protrusions (0.85% min À 1±0.13% min À 1 (Supplementary Fig. 9a–c). This observation is in line with a of observed MKs in Trpm7fl/fl-Pf4Cre mice versus report on resting foreskin fibroblasts in which NMMIIA and 0.05% min À 1±0.04% min À 1 in WT mice) that remained a-tubulin staining overlapped extensively at the cell cortex under attached to the cell body during the observation period resting, non-contractile conditions. However, on induction of cell (Fig. 2f,g; Supplementary Movies 1–3). migration and activation of NMMIIA, the overlap of NMMIIA The actin and microtubule cytoskeleton are critical for proper and a-tubulin staining decreased, allowing NMMIIA to exert its proplatelet formation2. We therefore analysed the cytoskeletal contractile effects on the actin cytoskeleton27. To further analyse architecture of Trpm7fl/fl-Pf4Cre MKs and found an increased the cause of the absent NMMIIA staining and the altered content and aberrant organization of microtubules in localization to the cell cortex in Trpm7fl/fl-Pf4Cre MKs in situ,we proplatelet-forming, resting and spread MKs (Fig. 2h; next allowed bone marrow-derived MKs to spread on a Supplementary Fig. 6a–c). In support of this, extraction of the collagen type I-coated surface. Surprisingly, on spreading of microtubule cytoskeleton of resting bone marrow-derived MKs Trpm7fl/fl-Pf4Cre MKs (Fig. 3d,e) or platelets (Supplementary by ultracentrifugation revealed an increased microtubule content Fig. 10a–d) we found a rapid degradation of NMMIIA that in the Triton X100 insoluble pellet fraction (Supplementary could be rescued by pretreatment with the NMMIIA Fig. 6d,e). Of note, increased microtubule stability is inhibitor blebbistatin or by Mg2 þ supplementation (Fig. 3e–g; associated with post-translational modifications of a-tubulin Supplementary Fig. 10e–h). Based on these findings we speculated 2 þ fl/fl-Pf4Cre that include detyrosination (Glu-tub) and acetylation (ac-tub) that deregulated [Mg ]i in Trpm7 cells may cause which could account for the increased number of microtubules in an increased activity of NMMIIA resulting in its rapid Trpm7fl/fl-Pf4Cre MKs (ref. 24). Nonetheless, we found an degradation on cell stimulation. In agreement with the increased prevalence of highly dynamic tyrosinated (Tyr) increased co-localization of NMMIIA to actin filaments in microtubules as evidenced by lower Glu-/Tyr- (2.5±0.3 resting Trpm7fl/fl-Pf4Cre platelets (Supplementary Fig. 9), for control versus 1.5±0.2 for Trpm7fl/fl-Pf4Cre MKs) and sedimentation of cross-linked actin filaments of resting platelets ac-/Tyr-tubulin ratios (2.2±0.2 for control versus 1.6±0.2 for revealed an increased amount of NMMIIA in the pellet fraction samples of Trpm7fl/fl-Pf4Cre mice), suggesting that accelerated (Supplementary Fig. 11a–c). On activation, NMMIIA efficiently microtubule assembly/dynamics accounted for the observed cross-linked actin filaments and accumulated in the pellet alterations (Supplementary Fig. 6d–g). Moreover, in line with fraction of WT platelets (Supplementary Fig. 11d–f). In sharp the critical role of microtubules in trafficking of intracellular contrast, we found a marked reduction in NMMIIA on cargo, electron microscopy revealed a non-homogeneous stimulation of Trpm7fl/fl-Pf4Cre platelets (Supplementary distribution of granules, tortuous membrane complexes Fig. 11d–f), similar to the findings in spread MKs (Fig. 3c) or and aberrantly sized proplatelets in mutant MKs (Fig. 2i; platelets (Supplementary Fig. 10a–d). To exclude that the Supplementary Fig. 7). Furthermore, we found thick and observed effects were due to the short observation period, bone densely packed proplatelets in bone marrow sinusoids with marrow-derived MKs were cultured for 48 h in the presence of signs of apoptosis reflecting impaired proplatelet fragmentation collagen types I or IV, two major components of the extracellular and release of preplatelets from Trpm7fl/fl-Pf4Cre MKs (Fig. 2f,j; matrix in the bone marrow. Strikingly, we found a reduced Supplementary Movies 2 and 3). In line with the impaired content of NMMIIA in Trpm7fl/fl-Pf4Cre MKs (reduction of proplatelet formation (Fig. 2e,g; Supplementary Fig. 5b and 39.9±10.3% for collagen I and 76.9±14.5% for collagen IV) Supplementary Movies 2 and 3), the recovery of the platelet count cultured in the presence of different collagens as compared with after antibody-induced platelet depletion was slightly delayed in control cells (Fig. 3h; Supplementary Fig. 12a,b). Together these Trpm7fl/fl-Pf4Cre mice (Supplementary Fig. 8). The pronounced results suggested an increased NMMIIA activity under resting increase above initial platelet counts might be attributed to conditions and that NMMIIA undergoes a rapid degradation on the increased number of MKs in Trpm7fl/fl-Pf4Cre mice and stimulation of platelets and MKs from Trpm7fl/fl-Pf4Cre mice thus alternative platelet release mechanisms as recently shown by allowing proplatelet formation and cell spreading. Nishimura et al.25.
2 þ Deregulated [Mg ]i perturbs NMMIIA activity.We Altered NMMIIA activity impairs proplatelet formation. hypothesized that deregulated Mg2 þ homeostasis26 and increased NMMIIA has been described as a downstream effector of TRPM7 NMMIIA activity may account for the impaired proplatelet kinase14,22 and importantly, abnormal function of NMMIIA has formation in Trpm7fl/fl-Pf4Cre MKs (refs 16,26). In support of been associated with impaired formation and fragmentation of this, either inhibition of NMMIIA activity or Mg2 þ proplatelets in humans and mice16. Moreover, Mg2 þ was supplementation could almost fully restore proplatelet formation recently shown to modify NMMIIA activity by regulating of Trpm7fl/fl-Pf4Cre MKs in vitro (Fig. 4a,b). Of note, while ADP release and its affinity to actin filaments, thus further pretreatment of foetal liver- (Fig. 4c) or bone marrow-derived MKs linking TPRM7 channel to NMMIIA function26. Strikingly, (Supplementary Fig. 13) with the Ca2 þ chelators 1,2-bis analysis of NMMIIA localization in MKs on whole-bone- (2-aminophenoxy) ethane-N,N,N0,N0-tetraacetic acid tetrakis marrow sections in situ revealed a homogeneous distribution in (acetoxymethyl ester) (BAPTA-AM) or ethylene glycol tetraacetic the cell body of control (92.1±2.0% in WT versus 14.7±1.1% in acid (EGTA) did not exert gross effects; non-specific chelation of Trpm7fl/fl-Pf4Cre mice), while it predominated in the cell cortex Ca2 þ and Mg2 þ with ethylenediaminetetraacetic acid (EDTA) of Trpm7fl/fl-Pf4Cre MKs (0.4±0.8% in WT versus 48.1±3.6% in significantly reduced proplatelet formation by MKs, suggesting that Trpm7fl/fl-Pf4Cre mice; Fig. 3a,b). A similar distribution pattern Mg2 þ is critical for this process28,29. According to a previous 8 2 þ 2 þ was found in vitro for foetal liver-derived MKs where in addition report ,Mg supplementation should restore [Mg ]i in mutant a significant accumulation of NMMIIA in proplatelets of mature MKs, which cannot be fully achieved through an upregulation of WT MKs was observed (Fig. 3c). Moreover, NMMIIA localization MagT1 expression under normal culture conditions (Fig. 4d,e).
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a b WT 100 *** Trpm7 fl/fl-Pf4Cre 80 60 *** 40 **
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NMMIIA WT 180 1st ab 130
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Figure 3 | Altered localization and accelerated degradation of NMMIIA in MKs. (a) Confocal microscopy images of immunostained bone marrow sections. Scale bars, 10 mm. MKs, proplatelets and platelets are identified by GPIb staining (green). NMMIIA is highlighted in cyan. Endoglin staining (red) labels vessels; 40,6-diamidino-2-phenylindole (DAPI) stains nuclei (grey). (b) Quantification of NMMIIA distribution in primary bone marrow MKs in situ. Cytopl, homogeneous cytoplasmic distribution; cortex þ cyotpl, homogeneous cytoplasmic distribution and some accumulation of NMMIIA at the cell cortex; cortex, accumulation of NMMIIA at the cell cortex; absent, no NMMIIA staining was detected. Values are mean±s.d. (n ¼ 5; 150 MKs). (c–g) Localization of NMMIIA (cyan) in proplatelet-forming foetal liver-derived MKs on day 4 of culture (c) and on collagen I (50 mgmlÀ 1) spread (3 h) bone marrow-derived MKs (d–g). The MK cytoskeleton was stained for a-tubulin (green) and F-actin (red). DAPI, blue. (e) Quantification of the relative NMMIIA content on confocal microscopy images of spread MKs. Values are mean±s.d. (n ¼ 5; 50 MKs per condition). Pretreatment of bone marrow-derived MKs with 25 mM blebbistatin (e,f) or 2.5 mM MgCl2 (e,g) prevented the degradation of NMMIIA (b) and restored its localization to podosomes. Scale bars, 10 mm(a,b). Scale bars, 25 mm(c,d). (h) Bone marrow-derived MKs of WT and Trpm7fl/fl-Pf4Cre mice were cultured for 48 h on a collagen I- or IV-coated (each 10 mgcmÀ 2) surfaces, lysed and NMMIIA prevalence was analysed by immunoblotting using two different antibodies (1st and 2nd ab). All images are representative of at least five animals. Unpaired Student’s t-test; ***,###Po0.001; **Po0.01.
Inhibition of NMMIIA restores cytoskeletal architecture. increased number of aberrantly organized microtubules in Besides increasing cortical tension through relaxation of Trpm7fl/fl-Pf4Cre platelets as compared with controls, similar to the underlying cytoskeleton17,30, blebbistatin treatment reduced findings in mutant MKs. This correlated with an increased the prevalence and stability of microtubules in proplatelet presence of highly dynamic Tyr-tubulin (151.4±3.7% of controls; protrusions, as evidenced by decreased Glu-/Tyr- (1.1±0.1 for Fig. 5e–g), leading to accelerated and uncontrolled microtubule WT and 1.3±0.1 for samples of Trpm7fl/fl-Pf4Cre mice) and ac-/ polymerization in mutant cells (Fig. 5a,h). Stable microtubules Tyr-tubulin ratios (1.3±0.1 for WT and 1.2±0.2 for samples of in Trpm7fl/fl-Pf4Cre platelets displayed a similar pattern of Trpm7fl/fl-Pf4Cre mice; Supplementary Fig. 14). Immunostaining post-translational modifications of a-tubulin (104.2±14.2% of (Fig. 5a–c) and electron microscopy (Fig. 5d) revealed an control for Glu-, and 97.4±9.4% of control for ac-tubulin) and
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a * b d Untreated 60 40 NS 200 *** 50 (kDa) WT KO 30 150 40 35 MagT1 30 20 100 20
10 expression 50 35 PPF MKs (%) 10 PPF MKs (%) Gapdh Relative MagT1 Relative 0 0 0 μ Untreated + 25 M blebbi + 2.5 mM MgCl2 c e *** + 2.5 mM MgCl 50 *** *** WT 200 NS 2 40 Trpm7 fl/fl-Pf4Cre 150 (kDa) WT KO 30 35 100 MagT1 20 ***
10 expression 50 PPF MKs (%) 35 0 MagT1 Relative 0 Gapdh Untr. 5 2.5 2.5 + 2.5 mM MgCl2 BAPTA EGTA EDTA (μM) (mM)
Figure 4 | Altered NMMIIA regulation accounts for the impaired proplatelet formation. (a–c) Pretreatment (90 min) of foetal liver-derived MKs with fl/fl-Pf4Cre 25 mM blebbistatin (a) or 2.5 mM MgCl2 (b) rescued proplatelet formation of Trpm7 MKs and increased those of WT controls. (c) Pretreatment (90 min) with the Ca2 þ chelators BAPTA-AM or EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N0,N0-tetraacetic acid) did not interfere with proplatelet formation whereas the non-selective cation chelator EDTA strongly impaired proplatelet formation in both WT and Trpm7fl/fl-Pf4Cre foetal liver- derived MKs. Values are mean±s.d. (n ¼ 6). (d,e) Densitometric analyses on immunoblots reveal a compensatory upregulation of MagT1 expression under normal culture conditions (d)inTrpm7fl/fl-Pf4Cre MKs that can be restored to the expression levels of WT controls by Mg2 þ supplementation (e). All images are representative of at least five animals. Values are mean±s.d. (n ¼ 5). Unpaired Student’s t-test; ***Po0.001; *Po0.05; NS, non-significant. were efficiently disassembled on cold challenge, thus excluding Further studies were focussed on the index case UCN 0012 enhanced stability as cause for the increased microtubule with a p.C721G (c.2161T4G) variant and pedigree members. content (Fig. 5e–h). Interestingly, pretreatment of WT platelets Sanger sequencing showed that the p.C721G variant was present with EDTA, but not EGTA, BAPTA-AM or MgCl2, mimicked in two further pedigree members with macrothrombocytopenia, these cytoskeletal alterations (Fig. 6a–c), changes that could also but was absent in one asymptomatic pedigree member, indicating be reverted by blebbistatin (Fig. 6d–f), thus further supporting the segregation with the TRPM7 genotype (Fig. 7a, Supplementary 2 þ notion that reduced [Mg ]i alters the subcellular localization Table 2 and Supplementary Fig. 17). For the fourth patient and activity of NMMIIA resulting in cytoskeletal disorganization. (pedigree member 2), now deceased, macrothrombocytopenia In agreement with this, we observed an increased content of was detected during her life (Fig. 7a). All other blood cell filamentous actin in resting Trpm7fl/fl-Pf4Cre platelets parameters and platelet function were normal for all (Supplementary Fig. 15a,b) and a decreased polymerization of p.C721G patients. Strikingly, paroxysmal atrial fibrillation was filamentous actin on platelet activation (Fig. 6g; Supplementary also present for the index case (pedigree member 5) and her Fig. 15b,c). Moreover, spread Trpm7fl/fl-Pf4Cre platelets displayed mother (pedigree member 2). an increased surface area (Fig. 6h; Supplementary Fig. 16) most Similarly to Trpm7fl/fl-Pf4Cre mouse platelets, we found a likely reflecting the activation-dependent rapid degradation of reduced content of Mg2 þ and an increased concentration of NMMIIA and consequently the loss of coherent cytoplasmic Ca2 þ in platelets from all tested patients with the p.C721G contractile forces normally generated by activated NMMIIA substitution as compared with healthy controls (Fig. 7b). Patch (refs 31,32). clamp studies on HEK293 cells confirmed that the p.C721G variant reduced TRPM7 channel activity by 85±4% as compared with WT controls (Fig. 7c) despite being localized to the cell Variants in TRPM7 may cause macrothrombocytopenia.We membrane (Supplementary Fig. 18). Likewise, in vitro studies on next hypothesized that some DNA variants of extreme low-fre- the p.R902C variant revealed, although less pronounced, a quency affecting TRPM7 channel function might also cause reduced TRPM7 channel activity by 39±6% (Supplementary macrothrombocytopenia in humans. Examining the results of Fig. 19). Platelets from all tested patients displayed an increased genome sequencing of 702 cases with bleeding and platelet dis- size, with a spherical shape and contained numerous orders of unknown genetic basis in the BRIDGE database of the vacuoles. Moreover, while platelets from controls displayed the NIHR BioResource—Rare Diseases revealed three cases with a typical discoid shape and microtubules organized into coils, the coding variant unobserved in nearly 81,000 control DNA sam- marginal band, platelets from the p.C721G patients showed an ples: UCN 0012 with p.C721G (c.2161T4G), UCN 0025 with aberrant distribution of granules and an increased number and p.R902C (c.2704C4T) variant, and UCN 0110 with p.G1353D anarchic organization of microtubules (Fig. 7d; Supplementary (c.4058G4A; Supplementary Table 1). Two of these three index Fig. 20). The abnormal cytoskeletal organization could be cases (p.C721G and p.R902C) showed low-platelet counts and confirmed by immunostaining on resting p.C721G platelets macrothrombocytopenia. Interestingly, the variant of index (Fig. 7e,f; Supplementary Figs 21 and 22) and similarly to the patient UCN 0110 (p.G1353D (c.4058G4A)) was Trpm7fl/fl-Pf4Cre platelets, which was not associated with increased located close to the a-kinase domain and the absence of microtubule stability (Fig. 7g; Supplementary Fig. 21). macrothrombocytopenia is in agreement with the results on the Trpm7KI mice (Supplementary Table 1 and Supplementary Fig. 2). Unfortunately, the family with the p.R902C variant was Altered regulation of NMMIIA in p.C721G platelets. Since unavailable for further studies. the ultrastructure of human platelets from individuals carrying
6 NATURE COMMUNICATIONS | 7:11097 | DOI: 10.1038/ncomms11097 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11097 ARTICLE
a b d WT Tr pm7 fl/fl-Pf4Cre 80 *** 60 40
WT 20 Platelets with
aberrant MT (%) 0 c α-tub F-act ) 6 2 *** m μ 4
2 fl/fl-Pf4Cre
MT surface ( 0
Tr pm7 WT Tr pm7 fl/fl-Pf4Cre
e f h 4 °C 4–37 °C WT KO WT KO 4 (kDa) PS PS PS PS T T T T 3 *** *** Tyr- 55 2 tub 1 *** WT Ratio (ac- : *** ** Ac- (a.u.) Tyr-tub) ** 0 55 tub PSPSTT α-tub F-act g 5 55 Glu- *** tub 4 *** *** 3 fl/fl-Pf4Cre Gapdh 2 *** 35 1 ** Ratio (Glu- : * Tyr-tub) (a.u.) Tyr-tub) Tr pm7 0 PSPSTT
Figure 5 | Altered cytoskeletal organization in Trpm7fl/fl-Pf4Cre platelets. (a) Confocal images of resting platelets. The platelet cytoskeleton was stained for a-tubulin (green) and F-actin (red). Scale bars, 3 mm. (b,c) Quantification of platelets with aberrant (b) microtubules (MT) and the MT surface per platelet (c). Values are mean±s.d. (n ¼ 5; 200 platelets). (d) Transmission electron microscopy (TEM) analysis of resting Trpm7fl/fl-Pf4Cre platelets revealed an anarchic organization of microtubules (arrow heads in inlays). Scale bar, 1 mm. (e) Tubulin cytoskeleton of resting platelets was isolated by ultracentrifugation of Triton X-100 lysates and immunoblotted to detect dynamic Tyr-tubulin and post-translational modifications of the microtubules by the analysis of acetylated (ac)- or detyrosinated (Glu)-tubulin. Gapdh served as loading control. Insoluble fraction (pellet, P); soluble fraction (supernatant, S); total protein (T). (f,g) Densitometry revealed a markedly reduced ratio of stable acetylated (f) and detyrosinated (g) microtubules to highly dynamic Tyr microtubules in Trpm7fl/fl-Pf4Cre platelets. Values are mean±s.d. (n ¼ 6) (h) Platelets were incubated for 3.5 h at 4 °C and if indicated rewarmed at 37 °C, fixed on PLL-coated slides and stained for F-actin (red) and a-tubulin (green). All images are representative of at least five animals. Unpaired Student’s t-test; ***Po0.001; **Po0.01; *Po0.05. the p.C721G variant strongly resembled that of Trpm7fl/fl-Pf4Cre likely in humans too. These results demonstrate a critical role of mouse platelets, we next analysed the distribution and stability of TRPM7-mediated Mg2 þ influx in regulating NMMIIA NMMIIA. Whereas in control platelets NMMIIA localized to the activity and cytoskeletal rearrangements during thrombopoiesis. marginal band, it was homogeneously distributed throughout the In support of this, it has been shown that Mg2 þ inhibits cytoplasm of platelets from the patients (Fig. 8a,b; Supplementary NMMIIA activity by reducing the ADP release rate and its affinity Fig. 22). In line with the observations on Trpm7fl/fl-Pf4Cre mouse for actin26. Furthermore, using Trpm7KI mice (Supplementary platelets, spread platelets from the patients showed an increased Fig. 2), we convincingly show that these effects occur surface area, a strong decrease in NMMIIA and an increased independently of TRPM7 a-kinase activity. However, it is too content of microtubules (Fig. 8c,d; Supplementary Figs 23 early to exclude that the kinase domain also acts as a molecular and 24). As for the mouse model, blebbistatin prevented loss of hub orchestrating yet unknown signalling events, and thereby NMMIIA on spreading (Fig. 8d,e; Supplementary Fig. 25) and contributes to the development of macrothrombocytopenia. rescued the cytoskeletal organization of resting platelets after cold The observed loss of NMMIIA in Trpm7fl/fl-Pf4Cre bone marrow challenge (Fig. 8f,g; Supplementary Fig. 26). As mentioned above, MKs in situ and in vitro on spreading could represent a patients 2 and 5 suffered from atrial fibrillation which might be physiological process to overcome the inhibitory effects of 2 þ associated with alterations in [Mg ]i (ref. 33). Furthermore, lack increased NMMIIA activity enabling proplatelet formation of TRPM7 has been associated with altered expression of (Fig. 3a,b,d–h; Supplementary Figs 10–12). In support of this channels, such as HCN4 encoding the pacemaker current in the hypothesis, NMMIIA degradation was most pronounced on conduction system of the heart that has also previously been culture of bone marrow-derived MKs on collagen type IV, which linked to atrial fibrillation20,34. predominates in the vascular niche where proplatelet formation occurs (Fig. 3a,b,h; Supplementary Fig. 12). Furthermore, using different antibodies and experimental approaches, we provide Discussion several independent lines of evidence that the observed loss of In the present study, we provide evidence that defects in TRPM7 NMMIIA on activation or spreading of TRPM7-deficient cells channel function cause macrothrombocytopenia in mice and represents protein degradation. However, it is important to note
NATURE COMMUNICATIONS | 7:11097 | DOI: 10.1038/ncomms11097 | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11097
ab d WT, 4–37 °C 100 4–37 °C + blebbi ØØ 80 *** *** 60 40
20 WT Platelets with Untreated
aberrant MT (%) 0 α-tub F-act
c )
1.0 2.5 2 6 α-tub F-act
m *** *** μ 4
2 fl/fl-Pf4Cre
MT surface ( 0 2.5 5.0 Untr. 1 2.5 2.5 5 1 2.5 1 2.5 Tr pm7 EGTA BAPTA MgCl2 EDTA M) μ (mM) (μM) (mM) WT e g fl/fl-Pf4Cre 80 *** 2.5 Tr pm7 BAPTA ( BAPTA *** *** *** 60 2.0 *** 1.0 2.5 1.5 40 1.0
(mM) EGTA (mM) 20 * 0.5 2 Platelets with act./rest.) (a.u.) MFI (Phal.-FITC aberrant MT (%) 0 0 Untr. Blebbi 10/1 0.01 1 0.1 MgCl A + U Thr CRP Rhd f h
) ## 1.0 2.5 * ### 2 3.0 160 Resting m
μ 120 2.0 + 25 μM blebbi 80 1.0
EDTA (mM) 40 per platelet (%) Rel. spreadRel. area MT surface ( 0 0 WT Tr pm7 fl/fl-Pf4Cre
2 þ Figure 6 | Deregulated [Mg ]i accounts for the altered cytoskeletal organization. (a) Resting control platelets were incubated for 3 h at 4 °Cin Tyrode’s-HEPES buffer supplemented with the indicated reagents. After rewarming to 37 °C platelets were fixed, allowed to adhere to PLL-coated coverslips and were stained for a-tubulin (green) and F-actin (red). (b,c) Quantification of microtubule (MT) morphology (b) and surface (c) revealed that decreasing 2 þ 2 þ fl/fl-Pf4Cre [Mg ]i (EDTA) but not [Ca ]i (EGTA or BAPTA) in WT platelets reproduces the cytoskeletal alterations found in Trpm7 platelets. Values are mean±s.d. (n ¼ 5; 200 platelets). (d–f) Rewarming of chilled and blebbistatin-pretreated (25 mM) Trpm7fl/fl-Pf4Cre platelets restored cytoskeletal organization. Scale bars, 3 mm. Values are mean±s.d. (n ¼ 6; 200 platelets). (g) The ratio of polymerized actin in activated versus resting platelets was determined. Platelet stimulation was achieved using A þ U, 10 mM ADP and 1 mM U46619; Thr, 0.01 U ml À 1 thrombin; CRP, 1 mgmlÀ 1 collagen-related peptide; Rhd, 0.1 mgmlÀ 1 rhodocytin. Values are mean±s.d. (n ¼ 6). (h) The relative spread surface area of untreated or blebbistatin-treated (25 mM) platelets was determined using F-actin staining as a measure. Values are mean±s.d. (n ¼ 6; 200 platelets). All images are representative of at least five animals. Unpaired Student’s t-test; ***,###Po0.001; ##oPo0.01; *Po0.05. that we cannot entirely exclude epitope masking due to with different genetic variants of TRPM7 are most likely modifications on NMMIIA (Figs 3 and 8; Supplementary due to the location and functional consequences of the Figs 10–12 and 23–25). mutations (Supplementary Table 1). While both the p.C721G It has recently been reported that plasma Thpo levels are also and the p.R902C variant are located in the channel domain regulated via Ashwell–Morell receptor-dependent clearance of and reduce channel activity (Fig. 7c and Supplementary Fig. 19), desialylated platelets, which in turn triggers expression of Thpo in the p.G1353D variant is not located within the channel hepatocytes and thus regulates platelet production35. However, it domain which may likely explain the distinct phenotype and is important to note that in agreement with the moderately absence of macrothrombocytopenia in the index patient UCN reduced platelet lifespan, we did not observe differences in 0025. Similar genotype–phenotype-relationships have been platelet terminal galactose levels suggesting that the decreased observed for patients suffering from MYH9-related plasma Thpo levels are reciprocally associated to the increased disorder38,39, Wiskott–Aldrich syndrome2,40 or filaminopathy number of MKs in the bone marrow and spleen (Fig. 2a,b; A41. Although we could not observe a dominant negative effect Supplementary Figs 3 and 4)36. Furthermore, the marked increase of the p.C721G variant on TRPM7 channel function in vitro, in platelet counts after immuno-depletion might result from an we speculate that the defect might be masked by the high- alternative platelet release mechanism as recently described by expression levels of TRPM7 in the used system or diminished Nishimura et al.25. channel activity may not represent the only mechanistic The here-described human disorder (Fig. 7; Supplementary explanation for the observed phenotypes. Furthermore, Tables 1 and 2) is clearly distinguishable from MYH9-related Trpm7 þ / À mice did not display macrothrombocytopenia platelet disease, which can be associated with hearing suggesting that gene haploinsufficiency does not account for the loss, cataracts or renal failure37. However, patients with variants observed disease (Supplementary Fig. 27). However, the impaired in TRPM7 and MYH9 both display macrothrombocytopenia channel activity and the striking phenotypic similarities in with more spherical platelets and partially, increased actomyosin platelets from patients and Trpm7fl/fl-Pf4Cre mice strongly contractility (Figs 7 and 8; Supplementary Figs 21–26)16. suggest that the variants in TRPM7 account for the observed The different clinical outcomes and symptoms of patients macrothrombocytopenia.
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a b c (nA) p.C721G 250 6 )
1.6 * –1 12 ) 200 –1 1.2 150 Mock 4 100 WT TRPM7 0.8 3 45 p.C721G 2 *** 50 +/M+/+ +/M 0.4 –100
Current (pA pF 0
Relative cation Relative (mV) content (pg fL 0 100 67 0 100 200 300 Mg2+ Ca2+ Time (s) –2 +/M g d e f 4 °C 4–37 °C )
2 8
V m μ 6 *** V 4 Control 2
α-tub F-act α-tub F-act MT surface ( 0 Patient 3 Patient Controls Patients Control Patient 3 Patient Patient 3Patient Control
Figure 7 | A human genetic variant in TRPM7 causes macrothrombocytopenia. (a) A heterozygous p.C721G variant of TRPM7 was identified by whole- exome sequencing in an index patient. Sanger sequencing confirmed that this variant (M) cosegregated with the macrothrombocytopenia (blue coloration) in the family pedigree. Open symbols indicate that no macrothrombocytopenia was observed. þ /M indicates that the family member was a carrier of the TRPM7 variant. þ / þ indicates that no variant was detected at that locus and no symbol that genotyping was not performed. (b) Total platelet cation content of healthy controls and patients 3, 5 and 6 was determined by inductively coupled mass spectrometry. Values are mean±s.d. (n ¼ 5 controls versus 3 patients). (c) Whole-cell patch clamp measurements on mock-transfected HEK293 (n ¼ 8), and cells overexpressing WT TRPM7 (n ¼ 13) or the p.C721G variant (n ¼ 13) revealed impaired channel activity. Measurements were conducted in absence of extracellular Mg2 þ to enhance current sizes. Currents were elicited by a ramp protocol from À 100 to þ 100 mV over 50 ms acquired at 0.5 Hz. Left panel: inward current amplitudes were extracted at À 80 mV, outward currents at þ 80 mV and plotted versus time of the experiment. Values are normalized to cell size as pA pF À 1 and represent mean±s.e.m. The depletion of intracellular Mg2 þ leads to the development of characteristic TRPM7-like currents in WT TRPM7 overexpressing HEK293 cells, whereas TRPM7 currents were abolished in mock-transfected, or p.C721G overexpressing HEK293 cells. Right panel: representative current–voltage relationshipsextractedat 250 s. WT TRPM7 overexpressing HEK293 cells show an I V À 1-relationship characteristic for TRPM7 (black trace), which are absent in mock-transfected (dashed black trace) or p.C721G overexpressing HEK293 cells (light grey trace). (d) Transmission electron microscopy (TEM) analysis of platelets reveals the abnormal platelet morphology associated with the mutation. V, vacuole. Scale bars, 1 mm. (e–g)Poly-L-lysine-immobilised resting (e) or cold-challenged (g) platelets were permeabilised and stained for F-actin (red) a-tubulin (green) and analysed by confocal microscopy. (f) Image analysis revealed an increased prevalence of microtubules (MT) under resting conditions. Values are mean±s.d. (n ¼ 3; 200 platelets). Unpaired Student’s t-test; ***Po0.001; *Po0.05.
Our study provides for the first time several independent lines with the SuperScript II reverse transcriptase (18064014, Invitrogen) according to 2 þ the manufacturer’s instructions. b-actin transcripts were determined as control. of evidence that proper regulation of Mg homeostasis in MKs 2 þ by TRPM7 plays a critical role in thrombopoiesis and platelet Primers used for the amplification of Mg transporters and channels are listed in Supplementary Table 3. sizing, both in humans and mice. Collectively, our results highlight the clinical need to carefully control platelet count and size in patients with deregulated Mg2 þ homeostasis. Mice. Conditional Trpm7-deficient mice were generated by intercrossing Trpm7fl/fl mice (exon 17 flanked by loxP sites) with mice carrying the Ultimately, our findings suggest, after careful consideration and Cre-recombinase under the platelet factor 4 (Pf4) promoter45 (Supplementary 2 þ assessment of adverse events, that Mg supplementation may Fig. 1b). Trpm7fl/fl mice were described earlier4, Pf4-Cre mice were kindly provided be used as a potential treatment of patients with increased activity by Dr Radek Skoda and Trpm7KI mice were generously provided by Dr Masayuki 6 of NMMIIA to manage thrombocytopenia. Despite the possibility Matsushita . All mice used in experiments were 12- to 16-week-old and sex-matched, if not stated otherwise. For experiments on MKs, only male of severe potential side effects in patients with impaired kidney animals were used. All animal experiments were approved by the district 2 þ function, Mg supplementation is considered as a relatively safe government of Lower Franconia (Bezirksregierung Unterfranken). Trpm7fl/fl-Pf4Cre therapeutic intervention42–44. Nevertheless, further studies in mice were genotyped via PCR on genomic DNA extracted from ear biopsies 0 0 animal models and patients are required to assess the efficacy and using the following primer pair: Trpm7KO-F: 5 -gaggtactggcaattgtgagc-3 and 2 þ Trpm7KO-R: 50-accacaaaatctctgccctct-30 yielding a 1,200-bp product for the safety of Mg supplementation in the management of certain floxed, 1000 bp product for the WT and a 400-bp fragment for the recombined cases of thrombocytopenia. allele (Supplementary Fig. 1c). For all experiments, the respective Trpm7fl/fl Finally, the fact that two of the patients in our study also (for Trpm7fl/fl-Pf4Cre mice) or Trpm7WT/WT (for Trpm7KI/KI mice) littermate suffered from paroxysmal atrial fibrillation promotes TRPM7 as a controls were used. All mice were derived from the following breeding strategies for Trpm7fl/fl X Trpm7fl/fl-Pf4Cre, yielding 50% Trpm7fl/fl WT and 50% novel candidate interfering with conductance in cardiac cells. Trpm7fl/fl-Pf4Cre mice or Trpm7WT/KI X Trpm7WT/KI resulting in 25% Trpm7WT/WT, 50% Trpm7WT/KI and 25% Trpm7KI/KI mice, respectively. Methods Semi-quantitative reverse transcription PCR. Total platelet RNA was isolated Platelet preparation. Mice were bled under isoflurane anaesthesia. Blood was after lysis using TRIzol reagent (15596018, Invitrogen) and tissue RNA was gained collected in heparin (20 U ml À 1, Ratiopharm) and centrifuged twice for 6 min at using the Qiagen RNeasy kit. To generate cDNA 1 mg RNA was reverse transcribed 300 g. Platelet-rich plasma (PRP) was supplemented with 2 mlmlÀ 1 of apyrase
NATURE COMMUNICATIONS | 7:11097 | DOI: 10.1038/ncomms11097 | www.nature.com/naturecommunications 9 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11097
a c e + 25 μM blebbi Control Control Control
NMMIIA Merge NMMIIA Merge NMMIIA Merge Patient 3 Patient Patient 3 Patient Patient 3 Patient
b d f g ### 8 40 Control Patient 3 ) 5 *** 2 *
max *** m
30 μ 4
) (a.u.) 6
–1 3 4 20 min 2 2 10 1 NMMIIA distrib. MFI (NMMIIA (% of cell volume) MT surface ( NMMIIA 0 0 0
Untr Blebbi 4–37 °C + blebbi α -tub F-act Controls Patients
Figure 8 | Altered NMMIIA activity accounts for the aberrant cytoskeletal organization. (a) Poly-L-lysine-immobilised resting platelets were permeabilized and stained for F-actin (red) a-tubulin (green), NMMIIA (cyan) and analysed by confocal microscopy. (b) Image analysis revealed an altered distribution of NMMIIA in platelets from patients with variants in TRPM7 as compared with healthy controls. Box plots display first and third quartiles and whiskers mark minimum and maximum values unless exceeding 1.5 Â interquartile range (IQR) of at least 70 platelets; symbols represent outliers and the horizontal line displays median (n ¼ 3 controls versus patient 3). Wilcoxon–Mann–Whitney test; ***Po0.001 (c–e) Degradation of NMMIIA on spreading of platelets from patient 3 (c,d) could be prevented by blebbistatin pretreatment (d,e). Values are mean±s.d. (n ¼ 3; 200 platelets). (f,g) Pretreatment of resting platelets from patient 3 with 25 mM blebbistatin restored cytoskeletal organization on cold challenge with subsequent rewarming (f), which was further evidenced by a significantly decreased surface covered by microtubules (MT) per platelet (g). Values are mean±s.d. (n ¼ 3; 200 platelets). Unpaired Student’s t-test (if not stated otherwise); ***,###Po0.001; *Po0.05.
À 1 À 1 (0.02 U ml ; A6410, Sigma-Aldrich) and 5 mlml prostacyclin I2 (PGI2) (10602400001, Roche) and allowed to spread for the indicated time. If indicated, (0.1 mgmlÀ 1; P6188, Sigma-Aldrich) and platelets were pelleted by centrifugation platelets were pre-incubated with toxins interfering with cytoskeletal dynamics for 5 min at 800 g, washed twice with Tyrodes-N-2-hydroxyethyl-piperazine-N02- such as colchicine (10 mM; A4082, AppliChem) before the spreading assay. After ethanesulfonic acid (HEPES) buffer (134 mM NaCl, 0.34 mM Na2HPO4, 2.9 mM the indicated time points, platelets were either fixed with 4% paraformaldehyde KCl, 12 mM NaHCO3, 5 mM HEPES, 5 mM glucose, 0.35% bovine serum albumin (PFA) in PBS and analysed or processed for immunofluorescence staining of the À 1 À 1 (BSA), pH 7.4) containing 2 mlml apyrase and 5 mlml PGI2. cytoskeleton. Therefore, platelets were fixed and permeabilized in PHEM buffer Blood samples of patients were obtained after informed consent in accordance (60 mM piperazine-N,N-bis-2- ethanesulfonic acid, 25 mM HEPES), 10 mM with the Declaration of Helsinki. Ethical approval was obtained from Inserm RBM EGTA, 2 mM MgCl2, pH 6.9) supplemented with 4% PFA and 1% IGEPAL CA- 04–14 for the project ‘Network on the inherited diseases of platelet function and 630, stained with anti-a-tubulin-Alexa F488 (3.33 mgmlÀ 1, 322588 (B-5-1-2), platelet production’ and by the Ethics Committee of the University Hospital Invitrogen), anti-NMMIIA (10 mgmlÀ 1, #3403, polyclonal, CellSignaling) and Wu¨rzburg. Fresh blood samples of patients and healthy volunteers were collected phalloidin-Atto647N (170 nM, 65906, Fluka) and mounted with Fluoroshield in 1/10 volume of acid-citrate-dextrose and centrifuged for 10 min at 200 g. PRP (F6182, Sigma-Aldrich). All used fluorophore-conjugated secondary antibodies was collected, supplemented with 2 ml of apyrase (0.02 U ml À 1, Sigma-Aldrich) were purchased from Invitrogen. Samples were visualized using a Leica TCS SP5 À 1 and 5 ml PGI2 (0.1 mgml , Sigma-Aldrich) per ml PRP. Before adherence to confocal microscope (Leica Microsystems). poly-L-lysine (PLL)-coated slides or spreading on different matrices, platelets were pelleted by centrifugation for 10 min at 800 g and washed twice with À 1 À 1 Tyrodes-HEPES buffer containing 2 mlml apyrase and 5 mlml PGI2. The Inductively coupled plasma mass spectrometry (ICP-MS). The cation content samples were allowed to rest for 30 min at 37 °C prior to being used in experiments. of 4 Â 107 platelets was analysed by ICP-MS on platelets from PRP. ICP-MS analysis was performed by ALS Scandinavia AB (Lulea, Sweden). Flow cytometry. Diluted blood (1:20) was incubated for 15 min at room m À 1 temperature (RT) with fluorophore-conjugated antibodies (2 gml ) directed Determination of platelet lifespan. The clearance of platelets from the circula- against platelet surface glycoproteins. Platelet count and size (FSC) were assessed tion was determined by the retro-orbital injection of 5 mg DyLight 488-labelled using a FACSCalibur (BD Biosciences) flow cytometer or with an automated blood anti-GPIX antibody2,47 in PBS into male mice. The percentage of labelled platelets cell analyser (Sysmex KX-21N). was determined by daily blood withdrawal and subsequent analysis by flow cytometry using fluorophore-conjugated platelet-specific antibodies2. Transmission electron microscopy of platelets. To analyse platelet ultrastructure, washed platelets were fixed with 2.5% glutaraldehyde (16,210, Electron Microscopy Sciences) in 50 mM cacodylate buffer (12,201, pH 7.2; AppliChem). After embedding Immunofluorescence staining on whole-femora cryosections. Femora of male in epon 812 (14,900, Electron Microscopy Sciences), ultrathin sections were gener- mice were isolated, fixed with 4% paraformaldehyde (A3813, AppliChem) and ated and stained with 2% uranyl acetate (22,400, Electron Microscopy Sciences) and 5 mM sucrose (S0389, Sigma-Aldrich), transferred into 10% sucrose in PBS and lead citrate (17,800, Electron Microscopy Sciences). Samples were visualized with an dehydrated using a graded sucrose series. Subsequently the samples were embedded EM900 transmission electron microscope (Carl Zeiss). in Cryo-Gel (39475237, Leica Biosystems) and shock frozen in liquid nitrogen. Frozen samples were stored at À 80 °C. Cryosections (7-mm-thick) were generated using the CryoJane tape transfer system (Leica Biosystems) and probed with Immunostaining on resting or spread platelets. Coverslips were either coated Alexa488-conjugated anti-glycoprotein (GP) Ib (ref. 48) antibodies (7A9, with PLL (Sigma-Aldrich), fibrinogen (100 mgmlÀ 1; F4883, Sigma-Aldrich) or 1.33 mgmlÀ 1), to specifically label platelets and MKs, and Alexa647-conjugated collagen-related peptide (CRP) (ref. 46; 6 mgmlÀ 1) overnight at 4 °C. For anti-CD105 antibodies (3.33 mgmlÀ 1, 120402 (MJ7/18), Biolegend) to stain the spreading on fibrinogen, platelets were stimulated with 0.01 U ml À 1 thrombin endothelium. Nuclei were stained using 40,6-diamidino-2-phenylindole
10 NATURE COMMUNICATIONS | 7:11097 | DOI: 10.1038/ncomms11097 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11097 ARTICLE
(1 mgmlÀ 1, D1306, Invitrogen). Samples were visualized with a Leica TCS SP5 Two-photon intravital microscopy of the bone marrow. Male and female mice confocal microscope (Leica Microsystems). were anaesthetized and a 1-cm incision was made along the midline to expose the frontoparietal skull while carefully avoiding damage to the bone tissue. The mouse was placed on a customized metal stage equipped with a stereotactic holder to Histology. Sections (3-mm-thick) of formalin-fixed paraffin embedded spleens and immobilize its head2,3. Bone marrow vasculature was visualized by injection of femora of male mice were prepared deparaffinized and stained with haematoxylin tetramethylrhodamine dextran (8 mg per g body weight, 2 MDa, Molecular Probes). (MHS32, Sigma-Aldrich) and eosin (318906, Sigma-Aldrich). MK number, Platelets and MKs were antibody-stained (0.6 mg per g body weight anti-GPIX- morphology and localization were analysed with an inverted Leica DMI 4000 B Alexa Fluor 488). Images were acquired with a fluorescence microscope equipped microscope. with a  20 water objective with a numerical aperture of 0.95 and a TriM Scope II multiphoton system (LaVision BioTec), controlled by ImSpector Pro-V380 software (LaVision BioTec). Emission was detected with HQ535/50-nm and In vitro differentiation and cultivation of bone marrow MKs. Haematopoietic ET605/70-nm filters. A tuneable broad-band Ti:Sa laser (Chameleon, Coherent) stem cells were isolated from male mouse bone marrow single-cell suspensions was used at 760 nm to capture Alexa Fluor 488 and rhodamine dextran using a magnetic bead-based negative depletion kit (anti-rat-IgG Dynabeads, fluorescence. ImageJ software (NIH) was used to generate movies. Invitrogen) in combination with rat-anti-mouse antibodies directed against CD45R/B220, TER-119, CD3, Ly-6G/C and CD11b (each 0.5 mg per 107 cells, 103216 (RA3-6B2), 116214 (TER-119), 100208 (17A2), 108414 (RB6-8C5) and Transmission electron microscopy of bone marrow MKs. For transmission 101214 (M1/70), Biolegend). Experiments were performed according to the electron microscopy of MKs, bone marrow was flushed from 12- to 16-week-old manufacturer’s protocol. Cells were cultured in MK-medium supplemented with male mice using Karnovsky fixative (2% PFA, 2.5% glutaraldehyde in 0.1 M À 1 50 mgml recombinant hirudin (Schering) at 37 °C, 5% CO2 for 3 days, before cacodylate buffer) and incubated overnight at 4 °C. Subsequently, fatty components MK enrichment using a two step BSA density gradient. On day 4, the percentage of of the samples were fixed with 2% osmium tetroxide in 50 mM sodium cacodylate proplatelet-forming MKs was determined using a light microscope (Zeiss). (pH 7.2), stained with 0.5% aqueous uranyl acetate, dehydrated with a graded ethanol series and embedded in Epon 812. Ultrathin sections were stained with 2% uranyl acetate (in 100% ethanol) followed by lead citrate. Images were taken on a MK spreading. In vitro cultivated bone marrow MKs of male mice were allowed to EM900 transmission electron microscope (Zeiss). adhere and spread at 37 °C and 5% CO2 on coverslips coated with fibrillar collagen type I (50 mgmlÀ 1; Nycomed), fibrinogen (100 mgmlÀ 1; F4883, Sigma-Aldrich) or CRP (6 mgmlÀ 1) for the indicated time. MK spreading was stopped by fixation and Platelet depletion. Circulating platelets were depleted in mice by injection of permeabilization of the cells using PHEM buffer supplemented with 4% PFA and 20 mg per 30 g body weight anti-GPIba-antibodies (Emfret, Eibelstadt, Germany) 1% IGEPAL CA-630. and platelet counts were monitored by flow cytometry for 10 days.
Immunofluorescence staining of cultured or spread MKs. To visualize the Immunoblot. Denatured resting or convulxin-stimulated (0.5 mgmlÀ 1) platelets or cytoskeleton, the cultured MKs were spun onto glass slides (Shandon Cytospin 4, untreated and MgCl2 supplemented MKs were lysed and separated by sodium Thermo Scientific), fixed and permeabilized in PHEM buffer supplemented with dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and blotted onto 4% PFA and 1% IGEPAL CA-630 and blocked with 1% BSA in PBS. F-actin was polyvinylidene difluoride membranes. NMMIIA (1 mgmlÀ 1, #3403, polyclonal, stained using phalloidin-Atto647N (170 nM, 65906, Fluka) and tubulin was stained CellSignaling; 1 mgmlÀ 1, MP3791; ECM Biosciences), Gapdh (1 mgmlÀ 1, G5262, with an anti-a-tubulin-Alexa F488 (3.33 mgmlÀ 1, 322588 (B-5-1-2), Invitrogen) Sigma-Aldrich) or MagT1 (10 mgmlÀ 1, AP5056a, Abgent) were probed with the antibody. WASp (10 mg ml À 1, #4860, CellSignaling) or NMMIIA (10 mgmlÀ 1, respective antibodies and detected using horseradish peroxidase-conjugated sec- #3403, polyclonal, CellSignaling) staining served as podosome marker. Nuclei were ondary antibodies (0.33 mgmlÀ 1) and enhanced chemiluminescence solution (JM- stained using 40,6-diamidino-2-phenylindole (1 mg ml À 1, D1306, Invitrogen) before K820-500, MoBiTec). Images were recorded using a MultiImage II FC Light mounting of samples with Fluoroshield (F6182, Sigma-Aldrich). Visualization was Cabinet (Alpha Innotech cooperation) device. Uncropped immunoblotting images performed with a Leica TCS SP5 confocal microscope (Leica Microsystems). are shown in Supplementary Figs 28 and 29.
Determination of MK ploidy. To determine bone marrow MK ploidy, both femora Extraction and sedimentation of the cytoskeleton. Resting or activated of male mice were isolated and the bone marrow was flushed and homogenized. (0.1 U ml À 1 thrombin (10602400001, Roche) platelets (5.7 Â 107) or MKs Non-specific binding sites of the 5D7 antibody were blocked by incubation of the cell (5 Â 106) were lysed in PHEM buffer containing 1% Triton X100, 6 mM taxol suspension with 0.02 mg ml À 1 anti-FcgR antibody (553142 (2.4G2), BD Pharmin- (A4667, AppliChem) for microtubule sedimentation or 2 mM phalloidin (A1488, gen). Afterwards, MKs were stained using a fluorescein isothiocyanate-conjugated AppliChem) for F/G-actin sedimentation and protease inhibitors (P8340, anti-CD41 antibody (10 mgmlÀ 1, 5D7). Finally, cells were fixed, permeabilized and Sigma-Aldrich). Polymerized and soluble fractions were separated by centrifuga- DNA was stained using 50 mgmlÀ 1 propidium iodide (P3561, Invitrogen) staining tion for 30 min at 100.000 g (microtubules) or 16.000 g (F/G-actin), respectively, solution with 100 mgmlÀ 1 RNaseA (EN0202, Fermentas) in PBS. Analysis was and 37 °C in a TLA-100 rotor (Beckman Coulter). Total platelet lysates, soluble performed by flow cytometry and FlowJo software (Tree Star Inc.). supernatants (S) and insoluble pellets (P) were supplemented with SDS–PAGE buffer containing 5% b-mercaptoethanol (M6250, Sigma-Aldrich). Samples were separated by SDS–PAGE, blotted onto polyvinylidene difluoride membranes and Plasma thrombopoietin levels. Plasma Thpo levels were determined using the probed with anti-Tyr-tubulin (0.5 mgmlÀ 1, MAB1864 (YL1/2), Milipore), anti- Mouse Thrombopoietin Quantikine ELISA Kit (DY488, R&D Systems). Briefly, acetylated tubulin (1 mgmlÀ 1, sc-23950 (6-11B-1), Santa Cruz Biotechnology Inc.), plasma was collected, diluted (1:5 in Reagent Diluent) and immediately applied as anti-detyrosinated-tubulin (1 mgmlÀ 1, AB3201, Milipore), NMMIIA (1 mgmlÀ 1, duplicates onto the anti-Thpo-IgG coated 96-well plate and incubated for 2 h at #3403, polyclonal, CellSignaling; 1 mgmlÀ 1, MP3791; ECM Biosciences) or b-actin room temperature. The plate was washed five times and incubated for 2 h with (1 mgmlÀ 1, SAB5500001 (SP124), Milipore) antibodies. 100 ml of horseradish peroxidase-conjugated anti-mouse Thpo antibodies. After another five washing steps, tetramethylbenzidine solution was added and incubated for 30 min at room temperature. The reaction was aborted by the addition of 100 ml Cold-induced microtubule disassembly. Microtubules were depolymerized by of diluted hydrochloric acid. Optical densities of the samples were determined incubation of platelets at 4 °C; reassembly was allowed by subsequent rewarming at using a Multiskan Ascent (96/384) plate reader (MTX Lab Systems) at 450 nm. 37 °C. Samples maintained at 4 °Cand37°C as well as rewarmed platelets were Wavelength correction was performed at 570 nm. fixed and permeabilized in PHEM buffer supplemented with 4% PFA and 1% IGEPAL CA-630 and allowed to adhere to a PLL-coated coverslips. Samples were À 1 immunostained using an anti-b1-tubulin (2.5 mgml , T4026, clone TUB 2.1, In vitro differentiation of foetal liver-derived MKs. The livers of 13.5–14.5-day- Sigma-Aldrich) or anti-a-tubulin Alexa F488 antibody (3.33 mgmlÀ 1, 322588 old female and male mouse foetuses were isolated from time-mated female mice (B-5-1-2), Invitrogen) and visualized using a Leica TCS SP5 confocal microscope and single-cell suspensions were prepared in MK-medium (IMDM medium (Leica Microsystems). containing 10% FCS, 1% penicillin/streptomycin (31980097, Gibco) and 50 ng ml À 1 recombinant Thpo (ref. 49). The homogenized foetal liver cells were cultured for 72 h at 37 °C and 5% CO2 and mature MKs were enriched on day three Actin polymerization. Washed platelets were incubated with a DyLight-649– of culturing using a BSA density gradient (3% and 1.5% BSA in PBS; A7030, labelled anti-GPIX antibody derivative (20 mgmlÀ 1). Subsequently, platelets were Sigma-Aldrich). If indicated the cells were treated with 25 mM blebbistatin (B0560, either left unstimulated or were stimulated with the indicated agonists for 2 min. Sigma-Aldrich) or maintained in culture medium supplemented with 10 mM Platelets were fixed with 0.55 volume of 10% paraformaldehyde in PHEM buffer MgCl2. On day 4, the percentage of proplatelet-forming MKs was determined by and treated with 0.1 volume 1% Triton X100. Subsequently, platelets were stained counting the total number of MKs as well as the number of proplatelet-forming with 10 mM phalloidin-fluorescein isothiocyanate (P5282, Sigma-Aldrich) for MKs under a light microscope (Zeiss). 30 min and analysed on a FACSCalibur.
NATURE COMMUNICATIONS | 7:11097 | DOI: 10.1038/ncomms11097 | www.nature.com/naturecommunications 11 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11097
Recruitment of patients. The index case and pedigree members were enrolled to a-tubulin staining of platelets and MKs using the same settings for both groups. the French ‘Network on the inherited diseases of platelet function and platelet NMMIIA distribution in resting platelets was analysed on three-dimensional production’ (INSERM RBM 01–14). Further subjects with bleeding and platelet surface plots and profile plots with the help of Fiji52. Means of the first and last disorders (BPD) and with unrelated rare disorders (control data) were enrolled to maxima and the mean between the first and last minima of the histograms were the BRIDGE-BPD project of the NIHR BioResource—Rare Diseases study determined and the NMMIIAmax to NMMIIAmin was calculated and depicted. (UK REC 13/EE/0325). Here, gene variants were retained in an analysis set if they NMMIIA distribution in spread MKs was analysed by grouping and counting the occur at a frequency of o1:10,000 in the exome aggregation consortium different distribution patterns. The surface of spread platelets was measured by database based on over 60,000 individuals (http://exac.broadinstitute.org/). All thresholding the F-actin staining. To analyse the distribution/content of NMMIIA cases provided informed written consent at enrolment. in spread platelets the area of the NMMIIA staining was correlated to the total cell size (F-actin) staining. The BRIDGE bleeding and platelet disorders collection. We searched for cases with coding variants of extreme low frequency in the TPRM7 gene by analysing Data analysis. The presented results are mean±s.d. from at least three data from 702 index cases with BPDs of unknown genetic basis recruited to the independent experiments per group, if not otherwise stated. Data distribution BRIDGE study of the NIHR BioResource—Rare Diseases. Gene variants were was analysed using the Shapiro–Wilk test and differences between control identified in genome sequencing data by comparison with reference human and knockout mice were statistically analysed using Student’s t-test or genome build GRCh37 and consequences were predicted using the Ensembl ver- Wilcoxon–Mann–Whitney test, respectively. P-valueso0.05 were considered sion 75 TPRM7 transcript ENST00000313478. Variants predicted to alter the as statistically significant *Po0.05; **Po0.01; ***Po0.001. Results with a amino acid sequence of the protein were retained in the analysis set if they P value40.05 were considered as not significant (NS). occurred at frequencies of o1 in 10,000 out of 67,000 individuals of the Exome Aggregation Consortium (ExAC) database, o1 in 1,000 in the 10,000 subjects of UK10K database and o1 in 100 in 3,453 subjects with unrelated rare disorders or References unaffected pedigree members recruited to other branches of the NIHR BioR- 1. Junt, T. et al. Dynamic visualization of thrombopoiesis within bone marrow. esource. We selected cases with extreme low-frequency variants and on the basis of Science 317, 1767–1770 (2007). clinical and laboratory characteristics, which were coded using Human Phenotype 2. Bender, M. et al. Megakaryocyte-specific Profilin1-deficiency alters microtubule Ontology (HPO) terms50 retrieved from the BRIDGE-BPD study database. We stability and causes a Wiskott-Aldrich syndrome-like platelet defect. Nat. then focused our further exploration on the cases with extremely rare variants that Commun. 5, 4746 (2014). were unobserved in the nearly 81,000 control DNA samples. The first step was to 3. Zhang, L. et al. A novel role of sphingosine 1-phosphate receptor S1pr1 in contact the families to explain the necessity to control these results by performing mouse thrombopoiesis. J. Exp. Med. 209, 2165–2181 (2012). co-segregation studies. One family met these requirements and additional family 4. Jin, J. et al. Deletion of Trpm7 disrupts embryonic development and members were examined in Paris at a French Centre for inherited platelet diseases. thymopoiesis without altering Mg2 þ homeostasis. Science 322, 756–760 (2008). 5. Ryazanova, L. V. et al. TRPM7 is essential for Mg(2 þ ) homeostasis in Co-segregation studies . Genomic DNA from peripheral blood mononuclear cells mammals. Nat. Commun. 1, 109 (2010). was extracted using a commercial DNA purification kit (Qiagen) and exon 17 of 6. Kaitsuka, T. et al. Inactivation of TRPM7 kinase activity does not impair its TRPM7 was amplified by polymerase chain reaction using the respective primer channel function in mice. Sci. Rep. 4, 5718 (2014). pairs: TRPM7_Ex17_forw: 50-ggagaatgtgctctggattc-30 and TRPM7_Ex17_rev: 7. Abed, E. & Moreau, R. Importance of melastatin-like transient receptor 50-gccaatcatccatcttgctc-30, expected product size 606 bp. PCR products were potential 7 and magnesium in the stimulation of osteoblast proliferation and purified with the help of QIAquick PCR purification kit (Qiagen) and processed for Sanger sequencing. migration by platelet-derived growth factor. Am. J. Physiol. Cell Physiol. 297, C360–C368 (2009). 8. Deason-Towne, F., Perraud, A. L. & Schmitz, C. The Mg2 þ transporter MagT1 Platelet function testing. Platelet function testing of human blood was performed partially rescues cell growth and Mg2 þ uptake in cells lacking the channel- by light transmission turbidometric aggregometry. To this end, PRP (2.5 Â 105 kinase TRPM7. FEBS Lett. 585, 2275–2278 (2011). platelet per ml) was isolated and stimulated with different concentrations of agonists 9. Schmitz, C. et al. Regulation of vertebrate cellular Mg2 þ homeostasis by (ADP, 5 and 10 mM; collagen I, 1 and 5 mg ml À 1; epinephrine, 5 mM; arachidonic TRPM7. Cell 114, 191–200 (2003). acid, 1 mM; ristocetin, 0.6 and 1.5 mg ml À 1; and TRAP (thrombin receptor 10. Su, L. T. et al. TRPM7 regulates polarized cell movements. Biochem. J. 434, activating peptide), 20 mM). Light transmission was followed over time with PPP 513–521 (2011). set as 100% light transmission. 11. Guilbert, A. et al. Transient receptor potential melastatin 7 is involved in oestrogen receptor-negative metastatic breast cancer cells migration through its kinase domain. Eur. J. Cancer 49, 3694–3707 (2013). Transient expression of WT and p.C721G TRPM7 variants. The TRPM7 12. Middelbeek, J. et al. TRPM7 is required for breast tumor cell metastasis. Cancer p.C721G and p.R902C variants were generated by site-directed mutagenesis on the Res. 72, 4250–4261 (2012). pcDNA3.1-TRPM7 expression construct51 using the QuickChange II XL site- directed mutagenesis kit (200517, Agilent Technologies) with the following primer 13. Liu, W. et al. TRPM7 regulates gastrulation during vertebrate embryogenesis. pairs: Trpm7_C721G_for: 50-ctggagtaattcaaccggcctcaagttagcgtttc-30 and Dev. Biol. 350, 348–357 (2011). Trpm7_C721G_rev: 50-gaaactgctaacttgaggccggttgaattactccag-30. The mutation was 14. Clark, K. et al. TRPM7 regulates myosin IIA filament stability and protein confirmed by sequencing. For transient expression of WT TRPM7 and the localization by heavy chain phosphorylation. J. Mol. Biol. 378, 790–803 (2008). p.C721G variant, human embryonic kidney (HEK) 293 cells were maintained at 15. Dorovkov, M. V. & Ryazanov, A. G. Phosphorylation of annexin I by TRPM7 channel-kinase. J. Biol. Chem. 279, 50643–50646 (2004). 37 °C and 5% CO2 in Earle’s minimal essential medium supplemented with 10% foetal bovine serum, 100 mgmlÀ 1 streptomycin and 100 U ml À 1 penicillin 16. Spinler, K. R., Shin, J. W., Lambert, M. P. & Discher, D. E. Myosin-II repression (Invitrogen). Cells were transiently co-transfected with eukaryotic expression favors pre/proplatelets but shear activation generates platelets and fails in vectors encoding WT TRPM7 or the p.C721G and p.R902C variant with 100 ng of macrothrombocytopenia. Blood 125, 525–533 (2015). an enhanced green fluorescent protein (EGFP) reporter construct (pcDNA3.1) 17. Shin, J. W., Swift, J., Spinler, K. R. & Discher, D. E. Myosin-II inhibition using Lipofectamine2000 (Invitrogen) according to the manufacturer’s instructions and soft 2D matrix maximize multinucleation and cellular projections typical and processed for patch clamp measurements or confocal microscopy. of platelet-producing megakaryocytes. Proc. Natl Acad. Sci. USA 108, 11458–11463 (2011). 18. Favier, R. & Raslova, H. Progress in understanding the diagnosis and molecular Electrophysiology. Patch clamp experiments were performed at a whole-cell genetics of macrothrombocytopenias. Br. J. Haematol. 170, 626–639 (2015). configuration. Currents were elicited by a ramp protocol from À 100 to þ 100 mV 19. Visser, D., Middelbeek, J., van Leeuwen, F. N. & Jalink, K. Function and over 50 ms acquired at 0.5 Hz and a holding potential of 0 mV. Inward currents regulation of the channel-kinase TRPM7 in health and disease. Eur. J. Cell Biol. were extracted at À 80 mV, outward currents at þ 80 mV and plotted versus time. À 1 93, 455–465 (2014). Data were normalized to cell size as pA pF . Capacitance was measured using the 20. Sah, R. et al. Timing of myocardial trpm7 deletion during cardiogenesis automated capacitance cancellation function of the EPC10 (HEKA). Nominally variably disrupts adult ventricular function, conduction, and repolarization. Mg2 þ -free extracellular solution contained 140 mM NaCl, 3 mM CaCl , 2.8 mM 2 Circulation 128, 101–114 (2013). KCl, 0 mM MgCl , 10 mM HEPES-NaOH, 11 mM glucose (pH 7.2, 300 mOsm). 2 21. Carter, R. N. et al. Molecular and electrophysiological characterization of Intracellular solution contained 120 mM Cs-glutamate, 8 mM NaCl, 1 mM MgCl , 2 transient receptor potential ion channels in the primary murine megakaryocyte. 10 mM HEPES, 10 mM BAPTA, 5 mM EDTA (pH 7.2, 300 mOsm). J. Physiol. 576, 151–162 (2006). 22. Clark, K. et al. TRPM7, a novel regulator of actomyosin contractility and cell Image analysis. All images of an experiment were acquired with the same laser adhesion. EMBO J. 25, 290–301 (2006). power, scan speed, detector settings, processed equally and analysed blinded. 23. Schachtner, H. et al. Megakaryocytes assemble podosomes that degrade matrix Microtubule surface area was determined with the help of Fiji52 by thresholding the and protrude through basement membrane. Blood 121, 2542–2552 (2013).
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24. Song, Y. & Brady, S. T. Post-translational modifications of tubulin: pathways to 49. Villeval, J. et al. High thrombopoietin production by hematopoietic cells functional diversity of microtubules. Trends Cell Biol. 25, 125–136 (2015). induces a fatal myeloproliferative syndrome in mice. Blood 90, 4369–4383 25. Nishimura, S. et al. IL-1alpha induces thrombopoiesis through megakaryocyte (1997). rupture in response to acute platelet needs. J. Cell Biol. 209, 453–466 (2015). 50. Westbury, S. K. et al. Human phenotype ontology annotation and cluster 26. Swenson, A. M. et al. Magnesium modulates actin binding and ADP release in analysis to unravel genetic defects in 707 cases with unexplained bleeding and myosin motors. J. Biol. Chem. 289, 23977–23991 (2014). platelet disorders. Genome Med. 7, 36 (2015). 27. Even-Ram, S. et al. Myosin IIA regulates cell motility and actomyosin- 51. Chubanov, V. et al. Disruption of TRPM6/TRPM7 complex formation by a microtubule crosstalk. Nat. Cell Biol. 9, 299–309 (2007). mutation in the TRPM6 gene causes hypomagnesemia with secondary 28. Meinders, M. et al. Sp1/Sp3 transcription factors regulate hallmarks of hypocalcemia. Proc. Natl Acad. Sci. USA 101, 2894–2899 (2004). megakaryocyte maturation and platelet formation and function. Blood 125, 52. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. 1957–1967 (2015). Nat. Methods 9, 676–682 (2012). 29. Larson, M. K. & Watson, S. P. Regulation of proplatelet formation and platelet release by integrin alpha IIb beta3. Blood 108, 1509–1514 (2006). Acknowledgements 30. Houk, A. R. et al. Membrane tension maintains cell polarity by confining We thank the patients and healthy volunteers for donating blood samples; Jonas Mu¨ller, signals to the leading edge during neutrophil migration. Cell 148, 175–188 Stefanie Hartmann and Birgit Midloch for excellent technical assistance. We are thankful (2012). to Prof David E. Clapham for providing the Trpm7fl/fl mice. We acknowledge 31. Cai, Y. et al. Nonmuscle myosin IIA-dependent force inhibits cell spreading Prof Willem Ouwehand, the NIHR BioResource—Rare Diseases and the associated and drives F-actin flow. Biophys. J. 91, 3907–3920 (2006). BRIDGE-BPD consortium for performing whole-exome sequencing of the index cases. 32. Leon, C. et al. Megakaryocyte-restricted MYH9 inactivation dramatically affects We thank the microscopy platform of the Bioimaging Centre (Rudolf Virchow Centre) hemostasis while preserving platelet aggregation and secretion. Blood 110, for providing technical infrastructure and support. This work was supported by the 3183–3191 (2007). Deutsche Forschungsgemeinschaft (SFB 688 to H.S., B.N. and A.B.). S.S. and J.M.M.v.E. 33. Shalev, H., Phillip, M., Galil, A., Carmi, R. & Landau, D. Clinical presentation were supported by a grant of the German Excellence Initiative to the Graduate School of and outcome in primary familial hypomagnesaemia. Arch. Dis. Child. 78, Life Sciences, University of Wu¨rzburg. V.C., S.Z. and T.G. were supported by the 127–130 (1998). Deutsche Forschungsgemeinschaft, TRR 152. Research in the Ouwehand laboratory was 34. Schulze-Bahr, E. et al. Pacemaker channel dysfunction in a patient with sinus supported by programme grants from the European Commission, NIHR and the BHF node disease. J. Clin. Invest. 111, 1537–1545 (2003). under numbers RP-PG-0310-1002 and RG/09/12/28096 and the laboratory also receives 35. Grozovsky, R. et al. The Ashwell-Morell receptor regulates hepatic funding from NHS Blood and Transplant. M.A.L. was supported by the Imperial College thrombopoietin production via JAK2-STAT3 signaling. Nat. Med. 21, 47–54 London Biomedical Research Centre. (2015). 36. Ng, A. P. et al. Mpl expression on megakaryocytes and platelets is dispensable for thrombopoiesis but essential to prevent myeloproliferation. Proc. Natl Acad. Author contributions Sci. USA 111, 5884–5889 (2014). S.S. designed the research, performed the experiments, analysed the data and wrote the 37. Seri, M. et al. Mutations in MYH9 result in the May-Hegglin anomaly, and manuscript. P.N. and R.F. performed the experiments, analysed the data and Fechtner and Sebastian syndromes. The May-Heggllin/Fechtner Syndrome contributed to the writing of the manuscript. M.F., S.F., S.K.G. and J.M.M.v.E. performed Consortium. Nat. Genet. 26, 103–105 (2000). the experiments and analysed the data. L.M. helped with the breeding of mice. H.S. 38. Althaus, K. & Greinacher, A. MYH9-related platelet disorders. Semin. Thromb. and A.T.N. helped with genetic analyses and commented on the manuscript. M.P.L. Hemost. 35, 189–203 (2009). collected the phenotype data. E.T. performed genetic analyses. S.B.-S. provided ethics 39. Chen, Y. et al. The abnormal proplatelet formation in MYH9-related support and helped with the blood withdrawals. M.M. provided Trpm7KI mice. P.B. macrothrombocytopenia results from an increased actomyosin contractility and analysed the data. S.Z. and V.C. performed the experiments, analysed the data and is rescued by myosin IIA inhibition. J. Thromb. Haemost. 11, 2163–2175 commented on the manuscript. M.A.L. co-established and co-directed the BRIDGE-BPD (2013). consortium and directed the genetic analysis by E.T. T.G. analysed the data and 40. Jin, Y. et al. Mutations of the Wiskott-Aldrich Syndrome Protein (WASP): commented on the manuscript. B.N. analysed the data and wrote the manuscript. hotspots, effect on transcription, and translation and phenotype/genotype A.B. supervised the research, performed the experiments, analysed the data and wrote correlation. Blood 104, 4010–4019 (2004). the manuscript. 41. Robertson, S. P. et al. Localized mutations in the gene encoding the cytoskeletal protein filamin A cause diverse malformations in humans. Nat. Genet. 33, 487–491 (2003). Additional information 42. Massy, Z. A. & Drueke, T. B. Magnesium and cardiovascular complications of Supplementary Information accompanies this paper at http://www.nature.com/ chronic kidney disease. Nat. Rev. Nephrol. 11, 432–442 (2015). naturecommunications 43. Shechter, M. Magnesium and cardiovascular system. Magnes. Res. 23, 60–72 (2010). Competing financial interests: The authors declare no competing financial interest. 44. Swaminathan, R. Magnesium metabolism and its disorders. Clin. Biochem. Rev. Reprints and permission information is available online at http://npg.nature.com/ 24, 47–66 (2003). reprintsandpermissions/ 45. Tiedt, R., Schomber, T., Hao-Shen, H. & Skoda, R. Pf4-Cre transgenic mice allow the generation of lineage-restricted gene knockouts for studying How to cite this article: Stritt, S. et al. Defects in TRPM7 channel function deregulate megakaryocyte and platelet function in vivo. Blood 109, 1503–1506 (2007). thrombopoiesis through altered cellular Mg2 þ homeostasis and cytoskeletal architecture. 46. Knight, C. et al. Collagen-platelet interaction: Gly-Pro-Hyp is uniquely specific Nat. Commun. 7:11097 doi: 10.1038/ncomms11097 (2016). for platelet Gp VI and mediates platelet activation by collagen. Cardiovasc. Res. 41, 450–457 (1999). 47. Nieswandt, B., Bergmeier, W., Rackebrandt, K., Gessner, J. E. & Zirngibl, H. This work is licensed under a Creative Commons Attribution 4.0 Identification of critical antigen-specific mechanisms in the development of International License. The images or other third party material in this immune thrombocytopenic purpura in mice. Blood 96, 2520–2527 (2000). article are included in the article’s Creative Commons license, unless indicated otherwise 48. Nieswandt, B., Bergmeier, W., Rackebrandt, K., Gessner, J. & Zirngibl, H. in the credit line; if the material is not included under the Creative Commons license, Identification of critical antigen-specific mechanisms in the development of users will need to obtain permission from the license holder to reproduce the material. immune thrombocytopenic purpura in mice. Blood 96, 2520–2527 (2000). To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
NATURE COMMUNICATIONS | 7:11097 | DOI: 10.1038/ncomms11097 | www.nature.com/naturecommunications 13